1328 PRINCIPLES OF PHYSICAL CHEMISTRY va uate «inorganic molecules ae bombarded with energetic electrons and thereby converted ino highly energetic posivey charged ions, called malar ions or parent ons, which can yderpo farther break up ito smal fons, called Fragment ions ox daugher Yong, Th Toss of a8 ceceon fro the formation of a molecular ion may Uns be represented as M—> M* i-ion, M°, may decompose to a pair of fragmems which could be either a radical plus ion, oa small molecule ps a adia-cton —— Ms mt tof om tof All the species—the molecular ions, the fragment ioas andthe fragment radical ions—are separated by defection na vacible mages el coring tothe carge rat. Te geet an ion caret atthe clletor, the mage of which depend nerve abundances. For ingly cared ts, he lower the mas, the moe easly isthe ion dette jn te magnetic id. We may meton hee hat gave molecles react on ela toate in 0 ways either an ecto is cape by the molecule giving radial sion oan ec sete fom he aneule giving art cto MMP oe MoMt 4 267 ‘The latter mode is far more probable and posiiveon ‘mass spectrometry is the result. Most ic molecules form molecular fons (M*) when bombarded with electron beam of energy 10-15 eV. However, electron bombardment energies ofthe oder of 70eV are generally employed fori is only at these energies that substantial fragmentation of molecules occurs, ‘The ions produced in the ionization chamber are separated in the magnetic analyzer on the of their m/e values. The kinetic energy, ofthe ion is given bythe expression E = Lm, where m is the mass of the ion traveling with velocity v. The molecular ioas generated ia the ionization chamber are expelled eletrostatially by means ofa low positive potential on a repeller plate (A) in the chamber. After emerging from the plate they are accelerated down the ion tube by avery high potential, V (of the order of several thousand vos) between plates B and C. When the ion is ‘repelled, its potential energy, 2¥, is converted into the kinetic energy. Thus, Wetmt oe Pe kvm (0 ‘When the ions are subjected to the magnetic fed, B. ofthe analyzer, they execute 2 citeulat ‘motion. At equilibrium, the centrifugal force of the ion (mv2/r) equals the centipeal force (eB) ‘exerted on it by the magnet, where ris the radius ofthe cizcular motion. Thus, mtr = aby 2) Hence, v= Brin 8) From Eqs. and 3, 2Vim = (eBrim)? 4 0 that mk = BAD ‘) Since B, V andr can be controlled experimentally, the ratio m/z can be determined, It should be noted that a dipostive ion of mass 54 gives rise tothe same m/z as a monopositive ion of mass 27. Under the experimental conditions employed here, most of te ions produced ar singly charged species When the ions pass through the detector, the signal, after being amplified (if it is of weak inteasy) is recorded. The most common amplification system used is te electron mater which Operates in a manner similar to the photomultper detector. Two essential featues of the recoding system in a mass spectrometer are that it must (a) have a very fast response and be able to scan several hundred peaks per second and (e) be able to raont peak intensities varying by a factor of ‘more than 10%. The anaog signa coming from de detector i fist converted to a digital form in an ‘nalog-to-dgital converter and the eigitalizd data ae tore! in computer memory, ASS SPECTROMETRY * 1329 In order to obtain the mas spectrum we have to determine the m/e catio for all fragments produced when a molecule is bombarded by highly energetic electrons as stated above, To do his, the detector set can be moved and the value ofr teasured for all particles continuously produces by tlectron bombardment inthe ion source. I is, Bowever, much simpler to vary B and V cotinvusly (Ga.3). This" way all partes eventually evel in a-sembcictle of fixed radius 1 The signal Inasty, which is directly proportional tthe munber of ions striking the detector, canbe ploted as 2 function of B or V, whichever is varied. In pace, however, i is convenient to measure a varying oven! so that B is often held constant. If B and rare constant, V is inversely proportional to m/z (Gq, 5) and mz can be ploced versus the signal intensity, giving te desired mass spetrum. Fig. 2. shows the mass spectrum of 2-netypentae (Cll). The ost abundant fon hsm vale of 48 catenin yf) swig tt he ot fo poi of re ezursetten Cy aC, ‘The most abindat fon (he Base pent) i arbitrarily assigned an abundance of 100; aloe intensities ate capresied as a petentge of is (lav ardances. The sal peak atm 86 is evenly he mela ion. The peaks atm 15, 29 and 71 coespond to CHC and Cy, respectively, et, The ragnent ‘ons arise from the cuptut ofthe molecular io, eter det or indie, wa a ” " . ge -CH,—}—CHy— Cts 2 0 a 8 MatB6 5, B° Cont 310 oe ae : : Bad sao few | ob eae Si 2 Spite! ma gon fee igh oi rn Hewes eure ras ae "The mass spectrum of a compound canbe obained on a smaller sample size (of the onfer of 1") than for any other spectrodpic technique. The principal disadvantage isthe destructive ature ofthe process, which precldes the sample recovery, the dificult of introducing very small ‘Samples into the high vaevum sytem needed for handling the ionic speci involved and the high cost Of the instruments. Modera mess spectrometers provide a wide cange of options in inlet systems, ion Sources, mass analyzes, detectors and signal processors. Batch inlet systems volatilize © sample enterally and allow it to Kak into the ionization region. The sample must have an appreciable vapour presse &elow about 500°C. The molecular ion (M) in the mass spectrum wsually has dhe highest m/z value, withthe exception of a characteristic group of peaks at m/t values of M'+1, M'42, MF4#3,..0 ete. The later are isotope peaks which arise from the fact that many of the cements present in organic molecules are not mono-isotopic. Peaks in the mass spectrum are usually sharp and appear at integral mass values (with the exception of those arising from some doubly ‘harged ins). Occasionally, peaks ae observed which are broad, spread over several mass units and fxe of low intensity. These ae called metastable peaks and give valuable information about the ‘mode of fragmentation ‘Most commercial mass spectrometers are equipped with accessories that allow use of several ion1330 PRINCIPLES OF PHYSICAL CHEMSTRY sources interchangeably. Some of these are Usted in Table 1 ‘TABLE t Soine Common Ton Soures ini Mas Sjectromeeis Nave acer | Te Toaiag Ager "Beswon inion EL | Gasp | errs cece Fillion HL | Gaspime | Hiphpotntlelecrode (Cec oizaion ct | caspase | Regt poste lan or eesron cpr at som bomtarnet ran | Deoptaa | Eoegeie sons Fil dsolon | Desrion | Hips peenil ence Laser desogion to | Deora | Laser beam Parma desorioo p> | desapioa | Highenergy Psion fapmens from Sr Seeonfary fon mass pecromey | sigs | Desrpion | 1 =20 eV irs Thermal on mss peromary | Tims | Desgdon | Hex Tira inition, ET, ws. av ad arr,» bam of mnoonegetie eens To nee ie tenes molecule, The desorption types ea sample robe from which energy dre tanstr ons Or moleules inthe condensed phase to ins in the gas pase Field foiation, Fl and field desorption, FD, employ large votes 10 profice lage electric ies (10? Viem) 10 joie toleules’ Gaseoos samples are eds he former and ono sales ona pro in the later. in chemical onan, Cl he spl is bord by postive ns produced by een torsbarnent at a seus stom or moccle fen CH) preset ne sein 100 to 10,000-fld exces. Less fragmentation results with CI than with El. In fast atom . tomberdmert, FAB, te simple i condesd ste fe — Spiel a ney oben it cs: (ec Macromolecules present a challénge for molar mass determination wsing mass spectrometry becayse it is difficult (0 produce gaseous ions'of large species wilt fragmento. However, two new techniques have emerged Jha circumvent this probiem. These are: matrix-assisted Liser desorption! ‘onizion (MALDD and electrospray ionization (ESI). We stall briefly discuss here MALDI-TOF mass spectrometry, s called because the MALDI tctinique is coupled to L—_—. time-o fght(TOF) ion-detector, Fig. 3 shows 2 schematic view of MALDI-TOF mass spectrometer. ‘The macromolecule is first embedded ii a solid matrix that often consists of an organic compound such 28 trans3-indoleacrylic acid and inorganic sls such as NaCl of silver triftuoroacetate. ‘A laser beam (usually a pulsed laser, such as Ny laser) ests macromolecules and ions from the solid matrix. The ionized macromolecules are acceeratot by an electrical potential difference (84) lover a distance d and then travel through adrift region of length. ‘The time, ¢, required for anion of mass m and charge number z to reach the detector a the end of the drift region is caleuated as follows : The kinetic energy ofthe ion is given by B= fro? = reaped ) where vis the speed of the ion, THe dit regon, J and the time of flight, fin the mass spectrometer fare both sulficietly short so that we can ignore acceleration and write v = Wt, Hence, substitution in Bq. 6 givés dee et Fig 3 The diagram of « MALDE-TOF mss miu? =2eaga O Rearrangement of Eq, 7 gives : nite a9 _ 8) pace MASS SPECTROMETRY 1331 Since d, Land Ap ace fixed for & given experiment, the time of ight, # ofthe ion is 2 dire measure of is m/z rao, which is given by . men desea ur ® “The mass spectroscopic tchalques of MACDI-TOF, ESI and'SLD (soft tase eourptiog), have many areas of application. Sophisticate biochemical analyses, which seemed imgosible only a few {ears ago, are now rottinely performed, thanks to these techaiques. MS(mass spectrometry) based Aralytoal methods are relatively cheap and are thus widely used in research laboratories around the ‘woul, The ESI and SLD techniques, developed, respectively, by Fenn and Tanaka, are standard techniques for structure analyses of peptides, proteins and carbohydrates. In the pharmaceutical industry, drug development bat undergone a radical change because of the new MS techniques. ESI aan SLD-based methods are now tsed inthe rap diagnosis of diseases such as malaria and ovarian, breast and prostate cancers. The 2002 Chemistry Nobel Prize was jointly awarded to the American chemist JB. Fenn, the Japanese cherist K. Tanaka and the Swiss chemist, K. Withrch "for identification and structae analyses of biological moleeues”, (Withrich used NMR spectroscopy for determining the theee dimensional srucute of biological macromoleculss.) [Djoamics of Plectron-Moléculé Collisions in Mass Spectrometry ‘We shall deal, in some deals, with the various processes that occur When energetic electrons ‘bombard a molecule. The exces electron energy ean be high enoug to break bonds inthe molecular ina, leading to its fragibentation. The aceleration potential ofthe bombarding election that is jst" ‘enough to initiate fragmentation is called the appearance potential of the fragment ion. AS the electfon ‘ombardzent energy becomes high, several bonds can undergo cleavage. Consider » hypothetical molecule BCDE bombarded with as electvoa. The following processes may occur in sequence (a) Ionization ; BCDE +e = —> —BCDE* + 2° £10) 1) Fragmentation of the Positive fon BCDE* —> BY + CDE. ay BCDE* —» BC* + DE. i 12) Bct’ —» Bt + Ce (13) « Bct + Be+Ct ° A BCDE* —» DE* + BC+ (15) DES —> Dt +E (16) « DEY >, Ds + BF 47) BCDE* —» BE* + CDs, ef. <8) BCDE* —>, CD* 4 BEs, etc. 9) (©) Pair Production : BODE +e —» BC+ + DE +e" 20) (@) Resonance Capture BCDE +e —> —BCDE" a) i can be easily visualised that only positively charged species ean travel tothe detector to give rise to peaks inthe mass spectrum. ‘Re above mentioned reactions are unimolecular decay processes. Fragment ions are generally formed in vibrationally excited slates that have-an excess of infernal energy. Sometimes, an ion, formed in an excited electronic state on the removal of a low-energy ion, undergoes internal conversion of energy, thereby producing an electronic ground state of the ion that has an excess of ‘bratlonal energy. In that eae the molecule can disocite in any of the excited states involved in